Method for the determination of body odor

ABSTRACT

A method for determining potential for body odor and a method for determining the effectiveness of a deodorant carried out by detecting a substance represented by the following formula (1) or a derivative thereof: 
                         
wherein R 1  is a hydrogen atom or a methyl group, R 2  is an alkyl group containing 1 to 5 carbon atoms, and R 3  is a hydrogen atom or a methyl group.

FIELD OF THE INVENTION

This invention relates to a method for determination of the possibilityof body odor generation, and in particular, apocrine odor generation.This invention also relates to a method for determination of theeffectiveness of a deodorant.

BACKGROUND OF THE INVENTION

With the recent increase in hygiene consciousness, an increasing numberof people are concerned about their body odor. The term “body odor” is ageneric term for the odor or smell generated in various parts of thebody, and main sources of the odor or smell include the head, mouth,armpit, groin, and foot sole. Among such sources of the odor or smell,the armpit smell is easily detected by the person himself/herself or bythe people in the immediate area of the person, and the factors such aspresence/absence, strength, and qualitative difference of the armpitsmell greatly affect the systemic body odor. Therefore, it is often thearmpit smell and not the body odor in general that is annoying for theperson himself/herself or for the people in the immediate area of theperson.

The armpit smell comprises a sour and stuffy smell and a smellspecifically found in the armpit. The sour and stuffy smell is generatedby decomposition of the eccrine sweat and grime secreted by the eccrinegland by the action of the indigenous bacteria of the skin. The maincause of this smell is lower carboxylic acids containing 2 to 5 carbonatoms, and this smell is called a “lower fatty acid smell”, or moresimply, an “acid smell”. In contrast, the smell specific to the armpitis generated by decomposition of apocrine sweat secreted from theapocrine gland containing proteins and cholesterol at a highconcentration by the action of the indigenous bacteria of the skin, andthis smell is called the “apocrine odor” or “armpit smell” (hereinafterreferred to as the “apocrine odor”).

Accordingly, for those people who are conscious about their body odor,and in particular, the armpit smell, it would be quite important to knowthe degree of their apocrine odor that they originally have in theirarmpit, and whether their own effort of reducing the body odor, forexample, by the use of deodorant products or surgical removal of theapocrine gland, actually had some effects.

Conventional methods that have been used in finding the degree (presenceor strength) of one's own apocrine odor in the armpit include (1) asensory test in which the armpit smell is evaluated by a third person,(2) an estimation based on information such as the presence of a personwith the armpit smell in his or her family and moist cerumen, and thelike which are said to be related to the apocrine odor.

However, the method (1) is associated with the difficulty of aquantitative assessment due to the large margin of the subjectivedecision of the panelist and assessment error due to olfactory fatigue.In the meanwhile, the method (2) does not directly assess the apocrineodor in the armpit, and this method cannot be used in an objective andquantitative determination of the effectiveness of the improvement ofthe armpit smell by the use of deodorant products or the effect of thesurgical removal of the apocrine gland.

Conventional methods that have been used to determine the effectivenessof the deodorant which is expected to suppress the apocrine odorgeneration include (1) a method in which the candidate deodorant isapplied to the armpit of the test subjects, and the smell of the armpitis evaluated after the application, and (2) a method in which thecandidate deodorant is mixed with the sweat collected from the armpit ofthe test subject, and the smell of the mixture is evaluated after anadequate incubation of the mixture.

Both of these methods, however, are associated with the considerableburden of the test subjects and this leads to the difficulty ofevaluating a large number of candidate deodorants under the sameconditions. In addition, quantitative assessment is difficult due to thelarge margin of the subjective decision of the panelist and assessmenterror due to olfactory fatigue. Furthermore, such method is associatedwith the difficulty in differentiating whether it was the generation ofthe apocrine odor that was suppressed by the candidate deodorant;whether the apocrine odor that had been generated was deodorized; orwhether the apocrine odor was sensed to a lower degree by the presenceof another odor. Accordingly, an accurate, efficient, objective, andquantitative determination of the “effectiveness in suppressing theapocrine odor generation” has been difficult.

The inventors of the present invention investigated odor components inthe human armpit, and found that the main components responsible for theapocrine odor are alcohol compounds having a thiol group at position 3(hereinafter sometimes referred to as 3-mercapto alcohol compound) astypically represented by 3-mercapto-3-methylhexane-1-ol,3-mercaptohexane-1-ol, 3-mercaptopentane-1-ol,3-mercapto-2-methylbutane-1-ol, and 3-mercapto-2-methylpentane-1-ol(Non-patent document 1).

The inventors of the present invention also found that3-mercapto-3-methylhexane-1-ol is present at a mass ratio of 10 or moretimes higher than that of other 3-mercapto alcohol compounds, and thatthis 3-mercapto-3-methyl-hexane-1-ol is the most common 3-mercaptoalcohol compound in the armpit of people having apocrine odor(Non-patent document 1).

The inventors of the present invention also found that the3-mercapto-3-methylhexane-1-ol is an optically active substancecomprising 72% by mass of (S)-3-mercapto-3-methylhexane-1-ol and 28% bymass of (R)-3-mercapto-3-methylhexane-1-ol (Non-patent document 1)

Patent Document 1 discloses that, as flavoring components,3-mercapto-2-methylbutane-1-ol has the characteristic odor like grass,leek, or gas, (S)-3-mercaptohexane-1-ol has a grass or rustic odor, and(R)-3-mercaptohexane-1-ol has odor like a grapefruit/passion fruit,gooseberry, or onion.

[Non-patent document 1] Japanese Journal of Taste and Smell Research,vol. 10, No. 3, pages 807-810 (December 2003).

[Patent Document 1] JP-A-2001-2634.

DISCLOSURE OF THE INVENTION

The present invention has enabled an objective and quantitativedetermination of the body odor, and in particular, latent apocrine odor,namely, the possibility (potential) of apocrine odor generation in thearmpit by detecting the substance represented by formula (1) asdescribed below. The present invention has enabled an objective andquantitative determination of the degree (presence/absence or strength)of apocrine odor by using the 3-mercapto alcohol compound as describedabove for the index.

The present invention has also enabled an accurate, efficient,objective, and quantitative determination of the effectiveness of adeodorant which suppresses generation of body odor, in particular, theapocrine odor in the armpit by quantitatively measuring the substancerepresented by formula (1) as described below.

Accordingly, this invention provides a method for determining potentialfor body odor carried out by detecting a substance represented by thefollowing formula (1):

or its derivative wherein R¹ is a hydrogen atom or methyl group and R²isan alkyl group containing 1 to 5 carbon atoms, and R 3is a hydrogen atomor methyl group.

This invention also provides a method for determining effectiveness of adeodorant carried out by quantitatively measuring a substancerepresented by formula (1).

The substance represented by formula (1) has an amino group and acarboxy group. The substance represented by formula (1) is believed tobe present in the interior or the exterior of the body in the state inwhich the amino group and/or the carboxy group has formed a salt, or inthe state with a higher molecular weight in which the amino group and/orthe carboxy group has formed a peptide bond with various amino acids, apeptide made of various amino acids, or a protein made of various aminoacids.

The present invention also provides a method for determining potentialof body odor by detecting a derivative of the substance represented byformula (1), namely, a derivative in which the amino group and/or thecarboxy group of such substance has formed a salt (for example, ahydrochloride or a metal salt), a derivative in which the amino groupand/or the carboxy group has formed a peptide bond with various aminoacids (for example, a dipeptide), or a derivative in which the aminogroup and/or the carboxy group has formed a peptide bond with an atomicgroup formed by condensation with dehydration of at least 2 molecules ofat least one member selected from various amino acids (for example, atripeptide, an oligopeptide, a polypeptide, or a protein). The presentinvention also provides a method for determining the effectiveness of adeodorant by quantitatively measuring such a derivative.

In the present invention, detection of the substance represented byformula (1) or its derivative at a high sensitivity have been enabled bychemically modifying the substance represented by formula (1) and/or itsderivative, in particular, by chemically modifying a functional group ofsuch substance and/or its derivative, and detecting the substancerepresented by formula (1) and/or its derivative which has beenchemically modified, and as a consequence, the present invention hasenabled to carry out the determination of the possibility (potential) ofgeneration of the apocrine odor and the determination of theeffectiveness of the deodorant at higher objectivity and quantitativeaccuracy.

The present invention also provides a method for determining potentialof the body odor or a method for determining effectiveness of adeodorant in which the determination carried out by detecting thesubstance represented by formula (1) having a chromophore which absorbsvisible or UV light or a chromophore which emits fluorescenceincorporated in at least one member selected from an amino group,carboxy group, and hydroxy group thereof. The present invention alsoprovides a method for determining potential of the body odor or a methodfor determining effectiveness of a deodorant in which the determinationcarried out by detecting the substance represented by formula (1) or itsderivative having a chromophore which absorbs visible or UV light or achromophore which emits fluorescence incorporated in at least onefunctional group thereof.

This invention also provides a substance represented by the followingformula (1a):

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows the result of a normal ion scan (Q3 scan) for the sweatcollected from a person with apocrine odor.

FIG. 1 b shows the result of the product ion scan (m/z=236) for thesweat collected from a person with apocrine odor.

FIG. 1 c shows the MS/MS spectrum for the peak at a retention time of4.25 minutes.

FIG. 2 a shows the result of a normal ion scan (Q3 scan) for the2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid (syntheticproduct).

FIG. 2 b shows the result of the product ion scan (m/z=236) for the2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid (syntheticproduct).

FIG. 2 c shows the MS/MS spectrum for the peak at a retention time of4.25 minutes.

FIG. 3 a shows the result of a normal ion scan (Q3 scan) for the sweatcollected from a person without apocrine odor.

FIG. 3 b shows the result of the product ion scan (m/z=236) for thesweat collected from a person without apocrine odor.

MODE FOR CARRYING OUT THE INVENTION

The inventors of the present invention made investigation on the methodfor objectively and quantitatively determining the possibility(potential) of apocrine odor generation in a state when the apocrineodor is not actually generated due to the effects of, for example, thesurrounding environment (season, temperature, moisture, etc.), namely,in a latent state of the apocrine odor (namely, as a state not yetmanifested).

The inventors of the present invention found, from the armpit of aperson with apocrine odor, the substance represented by formula (1) as aprevious stage substance (hereinafter also referred to as a “precursor”)from which 3-mercapto alcohol compound which is the main substanceresponsible for the apocrine odor is formed. This precursor itself hasno odor, and this is the significant difference of this precursor fromthe 3-mercapto alcohol compound which has a stinky beastly smell likethat of sulfur.

The inventors of the present invention also confirmed that the substancerepresented by formula (1) changes into the 3-mercapto alcohol compoundwhich is the main substance responsible for the apocrine odor by theaction of, for example, the microorganisms on the skin. The inventorsalso found that such 3-mercapto alcohol compound is present in thearmpit of a person having the apocrine smell, and that the amount of the3-mercapto alcohol compound is proportional to the strength of theapocrine odor.

The present invention relates to a method for objectively andquantitatively assessing the possibility (potential) of the armpit smellgeneration, and in particular, apocrine odor generation by detecting thesubstance represented by formula (1). The present invention also relatesto a method for accurately, effectively, objectively and quantitativelyassessing the effectiveness of a deodorant which is expected to suppressthe generation of the armpit smell, and in particular, apocrine odor.The present invention also relates to a novel substance represented byformula (1a).

The present invention has enabled an objective and quantitativeassessment of the body odor, in particular, the latent apocrine odor ofthe armpit, namely, the possibility (potential) of the apocrine odorgeneration by detecting the substance represented by formula (1) whichis the substance of a previous stage of the main substance responsiblefor the apocrine odor or a derivative of the substance represented byformula (1). The present invention has also enabled an accurate,efficient, objective, and quantitative assessment of the effectivenessof a deodorant which is expected to suppress the apocrine odorgeneration.

The substance represented by formula (1) and the apocrine odor in thehuman armpit has the relation as described below.

i) The substance represented by formula (1) is an odorless substancefound in the armpit of a person with the possibility (potential) ofgenerating apocrine odor.

ii) The substance represented by formula (1) changes into a 3-mercaptoalcohol compound which has a stinky and beastly smell like the smell ofsulfur and which is the main substance responsible for the apocrine odorby the action of, for example, microorganisms on the skin.

The 3-mercapto alcohol compound as mentioned above is an alcoholcompound which has a thiol group at position 3. Typical examples are3-mercapto-3-methylhexane-1-ol, 3-mercaptohexane-1-ol,3-mercaptopentane-1-ol, 3-mercapto-2-methylbutane-1-ol, and3-mercapto-2-methylpentane-1-ol, which are represented by the followingformula (2a) to (2e). These substances have the odor resembling apocrineodor.

The 3-mercapto alcohol compound detected from the armpit of a personwith apocrine odor is a substance formed by the decomposition of thesubstance represented by formula (1) by the action of, for example,microorganism on the skin.

It is estimated that the substances represented by formulae (2a) to (2e)are not the only substances present in the armpit of a person withapocrine odor, and substances having a similar chemical structure shouldalso be present in the armpit.

In addition, the amount of the 3-mercapto alcohol compound found in thearmpit of a person with apocrine odor is proportional to the strength ofthe apocrine odor, and this means that the 3-mercapto alcohol compoundis the main substance responsible for the apocrine odor of the humanarmpit.

Furthermore, in the armpit of a person with apocrine odor, the3-mercapto-3-methylhexane-1-ol represented by formula (2a) is found atan amount in terms of the mass ratio 10 times or higher than thesubstances represented by formula (2b) to (2e), and at a highestfrequency among such compounds in the armpit of a person with apocrineodor. In other words, the 3-mercapto-3-methyl-hexane-1-ol is the mostimportant substance responsible for the apocrine odor of the humanarmpit.

2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid represented bythe following formula (1a) is a novel substance which the inventors ofthe present invention found from the armpit of a person who may generateapocrine odor, and this substance is the substance of a previous stage(also referred to as the precursor) of the3-mercapto-3-methylhexane-1-ol which is the most important substanceresponsible for the apocrine odor.

The precursor represented by formula (1a) is itself an odorlesscompound, and in this point, this compound is greatly different from the3-mercapto-3-methylhexane-1-ol which has a stinky and beastly smell likesulfur. This precursor has the skeleton of3-mercapto-3-methylhexane-1-ol (2a) which is the most abundant3-mercapto alcohol compound in the armpit of a person with apocrine odorsimultaneously with the skeleton of an α-amino acid.

This precursor has been estimated to be a condensate of an odorantsubstance and an α-amino acid formed by removal of water (or H₂S) fromthe thiol group of the 3-mercapto-3-methylhexane-1-ol and hydroxy group(or thiol group) of serine (or cysteine).

This substance represented by formula (1a) changes to3-mercapto-3-methylhexane-1-ol (2a) which is the main substanceresponsible for the apocrine odor in the armpit of a person withapocrine odor by the action of, for example, the microorganisms on theskin.

Accordingly, among the substances represented by formula (1), the2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid represented byformula (1a) is particularly suitable as a substance for use indetermining the possibility (potential) of the apocrine odor generationin view of the relative abundance of this substance in the armpit of aperson having apocrine odor and the resulting ease of detection, and thefact that it changes to the 3-mercapto-3-methylhexane-1-ol which is thesubstance most responsible for the apocrine odor by the action of, forexample, microorganism on the skin. This substance has never beenreported not to mention the report of the isolation, and nevertheless,this substance is particularly suitable as a substance for use as anindex in the determination of the potential body odor. For the samereason, this substance is also particularly useful as a substance foruse as an index in the determination of the effectiveness of thesubstance which is expected for the deodorant effects of the armpitsmell, and in particular, apocrine odor.

The substances represented by formulae (2b) to (2e) which the inventorsof the present invention found from the armpit of the person havingapocrine odor are also estimated to be present as the substancesrepresented by the following formulae (1b) to (1e) in the stage beforeturning into the respective odorant substances. These precursors areestimated to change to the substances represented by formulae (2b) to(2e) by the action of, for example, microorganism on the skin.

It is estimated that the substances represented by formulae (1a) to (1e)are not the only substances present in the armpit of a person withapocrine odor, and substances having similar chemical structures mayalso be present in the armpit.

In the state when the substance represented by formula (1) is present inthe armpit but has not turned into the substance represented by formula(2) which is the main substance responsible for the apocrine odor, theapocrine odor can be regarded as latent (namely, not yet manifested).Accordingly, the detection of the substance represented by formula (1)carried out in the present invention enables objective and quantitativedetermination of the possibility (potential) of the apocrine odorgeneration. The measurement of the substance represented by formula (1)also enables objective and quantitative determination of theeffectiveness of the deodorant.

The 2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid representedby formula (1a) which is the substance which the inventors of thepresent invention found from the armpit of a person with apocrine odorhas an amino group and carboxy group.

Since the substance represented by formula (1) is an amino acidderivative, the amino group may be present in the form of —NH₂ or —NH₃⁺, and carboxy group may be present in the form of —COOH or —COO^(—)depending on the pH conditions.

The amino group in formula (1) is estimated to be present in theinterior and exterior of the body in the state of a salt (for example,hydrochloride); in the state bonded by a peptide bond to an α-amino acidselected from glycine, alanine, valine, leucine, isoleucine, proline,methionine, phenylalanine, tryptophan, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartic acid, glutamic acid,histidine, lysine, and arginine; or in the state bonded by a peptidebond to an atomic group formed by condensation with dehydration of atleast 2 molecules of at least 1 member selected from the amino acid asdescribed above.

The carboxy group in formula (1) is estimated to be present in theinterior and exterior of the body in the state of a salt (for example, ametal salt); in the state bonded by a peptide bond to the amino acid asdescribed above; or in the state bonded by a peptide bond to an atomicgroup formed by condensation with dehydration of at least 2 molecules ofat least 1 member selected from the amino acid as described above.

In other words, the substance represented by formula (1) is estimated tobe present with its amino group and carboxy group moieties forming asalt or bound to another peptide or protein.

Accordingly, in the present invention, the determination of thepossibility (potential) of the apocrine odor generation can also berealized by detecting a derivative of the substance represented byformula (1) at the amino group, for example, a salt (for example,hydrochloride); a substance bonded by a peptide bond to an α-amino acid(for example, a dipeptide) selected from glycine, alanine, valine,leucine, isoleucine, proline, methionine, phenylalanine, tryptophan,serine, threonine, cysteine, tyrosine, asparagine, glutamine, asparticacid, glutamic acid, histidine, lysine, and arginine; or a substancebonded by a peptide bond to an atomic group formed by condensation withdehydration of at least 2 molecules of at least 1 member selected fromthe amino acid as described above (for example, tripeptide,oligopeptide, polypeptide, and protein). The objective and quantitativedetermination of the effectiveness of the deodorant can also be realizedby quantitatively determining a derivative of the substance representedby formula (1) at the amino group.

In the present invention, the determination of the possibility(potential) of the apocrine odor generation can also be realized bydetecting a derivative of the substance represented by formula (1) atthe carboxy group, for example, a salt (for example, a metal salt); asubstance bonded by a peptide bond to the amino acid as described above(for example, a dipeptide); or a substance bonded by a peptide bond toan atomic group formed by condensation with dehydration of at least 2molecules of at least 1 member selected from the amino acid as describedabove (for example, tripeptide, oligopeptide, polypeptide, and protein).The objective and quantitative determination of the effectiveness of thedeodorant can also be realized by quantitatively determining aderivative of the substance represented by formula (1) at the carboxygroup.

Accordingly, the derivative of the substance represented by formula (1)which is detected or quantitated in the present invention may includethe derivatives as described above.

In the present invention, the body odor may be determined by furtherdetecting the substance represented by formula (2) in addition to thesubstance represented by formula (1) or its derivative.

wherein R¹, R², and R³ are as defined above.

Typical examples of the substance represented by formula (2) include thesubstances represented by formulae (2a) to (2e). These substances aregenerated by the action of, for example, microorganisms on the skin, andthese are the main substances that cause apocrine odor.

Accordingly, a more comprehensive, objective, and quantitativedetermination of the possibility (potential) of the armpit smellgeneration and the degree (presence/absence, strength, etc.) of theapocrine odor which has already been generated can be accomplished bydetecting the substance represented by formula (2) which is the mainsubstance responsible for the apocrine odor in addition to the substancerepresented by formula (1).

The present invention is also capable of determining the body odor bydetecting a substance represented by the following formula (3), (4),(5), or (6).

wherein R¹, R², and R³ are as defined above.

The substances represented by formulae (5) and (6) are the substancesproduced by decomposition of the substances represented by formulae (3)and (4), respectively, by the action of, for example, microorganisms onthe skin, and these are the substances that cause apocrine odor.

Accordingly, latent apocrine odor, namely, the possibility (orpotential) of the generation of apocrine odor can be objectively andquantitatively determined by further detecting the substance representedby formula (3) or (4), which is the substance before conversion into thesubstance represented by formula (5) or (6) that is responsible for theapocrine odor.

Furthermore, simultaneous detection of the substance represented byformula (5) or (6) that is responsible for the apocrine odor will enablean integral, objective, and quantitative determination of thepossibility (or potential) of the generation of the apocrine odortogether with the degree (presence/absence, strength, etc.) of theapocrine odor that has already been generated.

Next, the method of determining body odor by detecting a substancerepresented by any of formulae (1) to (6) and/or a derivative thereof isdescribed in further detail by referring to typical embodiments.

The method for determination of the body odor according to the presentinvention is not particularly limited. The determination, however, maybe accomplished by analyzing whether the substance of any one offormulae (1) to (6) and/or its derivative is present in the armpit byvarious chemical, physical, biological, or other analytical method, anddetermining the body odor based on the result of the analysis.

For example, determination of the body odor may be conducted by (1)collecting the substance present in the armpit, and (2) qualitativelyanalyzing whether there is a substance represented by any of formulae(1) to (6) and/or its derivative in the collected substance, and whenthe substance represented by any of formulae (1) to (6) and/or itsderivative is present, quantitatively analyzing the amount of suchsubstance to thereby determine the body odor based on the comprehensiveresult of the analysis.

The substance in the armpit can be collected, for example, by rubbingthe armpit with a fabric such as absorption wadding, by spraying thearmpit with physiological saline or the like and then wiping the armpitwith a fabric such as absorption wadding, by sandwiching a cotton padbetween the armpit for a predetermined period, or by wearing anunderwear or a T shirt having a cotton pad fixed to the part that comesin contact with the armpit for a predetermined time, and such method maybe used either alone or in combination of two or more methods.

Exemplary methods used for promoting the sweating in the armpit includeapplication of exercise stress such as running and muscular training,application of temperature stress by entering a high temperature roomsuch as a sauna, application of mental stress through mental arithmeticor viewing a horror movie, application or injection of a bloodcirculation accelerator such as pepper extract or sweat gland stimulantsuch as epinephrine on or in the skin of the armpit, and such method maybe used either alone or in combination of two or more methods.

If desired, the method for collecting the substance in the armpit andthe method for promoting sweating in the armpit can be used incombination.

In the present invention, the analyte collected from the armpit by themethod as described above may be treated by various purification orconcentration techniques using the difference in chemical and otherproperties of the substance represented by formula (1) from othersubstances to thereby facilitate detection of the substance representedby formula (1).

For example, the substance represented by formula (1) has acharacteristic feature that it is readily soluble in water but not inoil, and therefore, oil soluble contents in the analyte may be separatedby using the difference in solubility in water or oil to therebyincrease the concentration of the substance represented by formula (1)in the analyte.

More specifically, oil soluble contents from sebum such as triglyceridesand higher fatty esters can be removed from the analyte collected by thecollection methods of the substances in the armpit as described above byadding an organic solvent such as hexane, pentane, dichloromethane, ordiethylether to the analyte and thoroughly stirring the mixture tothereby dissolve the oil-soluble content in the analyte in the organicsolvent, separating the mixture into the aqueous layer and the organiclayer by such means as centrifugation, and removing the oil-solublecontent from the analyte together with the solvent to thereby facilitatedetection of the substance represented by formula (1).

In the present invention, any of the substances represented by formulae(1) to (6) may be synthesized by chemical or other method, and the thussynthesized substance may be used for the standard.

As described in the following section, the substances represented byformulae (1) to (6) and/or their derivatives are substances that can beproduced by chemical synthesis, and synthetic products with constantquality can be supplied in a stable manner.

For example, the substances represented by formulae (1) and (2) can besynthesized by the following reaction scheme (7). In this reactionscheme, a fatty ester derivative (a) having an unsaturated structure isused for the starting material, and benzyl mercaptan or the like isintroduced to this fatty ester derivative (a) by addition to therebyproduce a fatty ester derivative (b) having a thioether structure atposition (3). This fatty ester derivative (b) is reduced by a reducingagent such as lithium aluminium hydride to produce an alcohol derivative(c) having a thioether structure at position 3.

The 3-mercapto alcohol compound represented by formula (2) issynthesized when a benzyl group is subsequently eliminated by Birchreduction. This 3-mercapto alcohol compound and 3-chloroalanine havingits amino group protected with a tert-butoxycarbonyl group (BOC group)are condensed to produce an α-amino acid derivative (d) having athioether structure, and the product is further treated with an acid(trifluoroacetic acid, etc) to eliminate the protective group. Thesubstance represented by formula (1) is thus synthesized.

wherein R¹, R², and R³ are as defined above, R⁴ is an alkyl group, andR⁵ is benzyl group.

The substance represented by formula (1) has an α-amino acid skeleton.This substance is a component found from the human armpit, andconfiguration of the α carbon is an L-configuration. However, when thesubstance represented by formula (1) is used in the body odordetermination by chemically synthesizing such substance, the substanceis not particularly limited for its configuration, and an amino acid ofD configuration and an amino acid mixture containing the amino acids ofboth L and D configurations at any ratio can be used in addition to theamino acid of L configuration. For example, the 3-chloroalanine used inthe synthesis as mentioned above may be any of L-3-chloroalanine,D-3-chloroalanine, DL-3-chloroalanine, or a 3-chloroalanine mixturecontaining any of these 3-chloroalanines at an arbitrary ratio.

The substance represented by formula (1) also has the skeleton of3-mercapto-3-methylhexane-1-ol which is the main substance responsiblefor the apocrine odor.

The 3-mercapto-3-methylhexane-1-ol which is the main substanceresponsible for the apocrine odor is an optically active substancecontaining 72% by mass of (S)-3-mercapto-3-methylhexane-1-ol and 28% bymass of (R)-3-mercapto-3-methylhexane-1-ol. However, when the substancerepresented by formula (2) is chemically synthesized for body odordetermination, the composition is not particularly limited, and it ispossible to use the (S) or (R) form produced by selective synthesis bythe asymmetric synthesis technique using an asymmetric catalyst such astransition metal or a mixture of such selectively synthesized forms atan arbitrary ratio.

Alternatively, a racemic body comprising 50% by mass of the (S) form and50% by mass of the (R) may be synthesized and used with no furthertreatment, or after separating the (S) and (R) forms by any of thephysical, chemical, biological or other technique such ascrystallization or chromatography. When the (S) and (R) forms areseparated, they may be used alone or after mixing at an arbitrary ratio.

Similarly, when the substance represented by formula (1) is produced bychemical synthesis, the substance represented by formula (2), namely,3-mercapto-3-methylhexane-1-ol used for the starting material is notparticularly limited for its configuration, and either one of (S) and(R) forms or a mixture of (S) and (R) forms at an arbitrary ratio can beused.

Since the substance represented by formula (1) has two asymmetric carbonatoms, as much as 4 isomers are present. The substance represented byformula (1) is not particularly limited for such configuration, and anas-synthesized product which is a mixture of such isomers at a certainratio can be used either with no further processing or after separatinginto the isomers by various separation and purification techniques. Thethus separated isomers may be used either alone or as a mixture of twoor more at an arbitrary ratio.

In the determination of the body odor by detecting the substancesrepresented by formulae (1) to (6), the determination can be carried outeither by detecting such substance itself or by detecting a derivativeof such substance. The determination may also be carried out bydetecting both the substance itself and a derivative thereof.

The method used in the present invention in qualitatively and/orquantitatively assaying whether the substance collected from the armpitcontains any of the substances represented by formulae (1) to (6) and/orits derivative is not particularly limited. Exemplary techniques includeanalysis by chromatographic separation and immunochemical assay (alsoreferred to as “immunoassay”) using, for example, a monoclonal antibody,and these techniques may be combined with various techniques such aschemiluminescence, chemical amplification, and electrochemicalmeasurements, which are capable of realizing amplification orsupersensitivity in the stage of the detection.

The chromatographic processes which may be used in the present inventionare not particularly limited, and examples include liquidchromatography, gas chromatography, thin layer chromatography, andelectrophoresis.

Liquid chromatography is a process wherein the solute is separated bythe difference in the affinity for the stationary phase and the mobilephase, and the present invention can make use of various separationmechanisms (interaction between the elutes and the stationary phase).Examples include adsorption chromatography, partition chromatography,ion exchange chromatography, and size exclusion chromatography (gelfiltration chromatography or gel permeation chromatography).

More specifically, in the adsorption column chromatography, for example,the separation is accomplished by the difference in the adsorptionfactor when the solute is adsorbed by the stationary phase through aphysical or chemical process, and the components are separated from eachother in the column and retained for the time inherent to each component(this time is called “retention time”) and then eluted from the columnto pass through the detector in the exterior of the column. In the caseof the size exclusion chromatography, the separation is accomplished bythe difference in the degree of exclusion or permeation of the elutemolecules in the three dimensional network structure depending on thesize of the solute molecule.

The presence of any of the substances represented by formulae (1) to (6)and/or their derivatives in the substance collected from the armpit canbe qualitatively assayed by the retention time read from thechromatogram of the synthetic product and the human sample and thespectral peak information of the chromatogram obtained from thedetectors.

For example, when a single mass spectrometer (MS) or two or moremutually connected mass spectrometers are used as the detector of achromatographic process, mass spectral information (also referred to as“fragmentation pattern” or the “cleavage pattern”) based on molecularweight and molecular structure for each of the components separated onthe column can be obtained. Since the mass spectral information isinherent to each substance, the presence of any of the substancesrepresented by formulae (1) to (6) and/or their derivatives in thesubstance collected from the armpit can be qualitatively assayed byusing such mass spectral information.

When the detector used in the chromatography is one member or two ormore connected members selected from visible and UV absorptiometer(visible/UV), infrared spectrometer (IR), Raman spectrometer, andnuclear magnetic resonance spectrometer (NMR), visible and UV (Vis/UV)absorption spectrum, infrared (IR) spectrum, Raman spectrum, and nuclearmagnetic resonance (NMR) spectrum inherent to the molecular structure ofeach component are obtained from the components separated on the column.Since the spectral information is unique to each substance, the presenceof any of the substances represented by formulae (1) to (6) and/or theirderivatives in the substance collected from the armpit can bequalitatively assayed.

In the chromatographic separation and assay system, size (area) of thepeak in the chromatogram is proportional to the concentration of thesubstance corresponding to the peak. Accordingly, when any of thesubstances represented by formulae (1) to (6) and/or their derivativesis present, the content of such substance can be quantitatively assayedby using the area of the corresponding peak in the chromatogram as areference.

When the synthesized product of the substance represented by formula (1)is used for the standard, calibration curves in various concentrationranges can be prepared by using the thus prepared standard, and theamount of the substance represented by formula (1) in the collectedsample can be calculated by using the thus depicted calibration curves.

In the chromatographic separation and assay system, not only thequalitative assay of the presence of any of the substances representedby formulae (1) to (6) and/or their derivatives in the substancecollected from the armpit, but also the quantitative assay of the amountof such substance can be conducted by using the size (area) of the peakin the chromatogram for reference. The body odor can then be determinedon the basis of the comprehensive result of the analysis.

When a qualitative assay for the presence of any of the substancesrepresented by formulae (1) to (6) and/or their derivatives in thesubstance collected from the armpit or a quantitative assay for theamount of such substance is conducted by using the chromatographicseparation and assay system, the substances represented by formulae (1)to (6) and/or their derivatives maybe chemically modified in order toimprove selectivity and detection sensitivity of the assay, and the bodyodor can be determined by detecting the chemically modified substance.The body odor can also be determined by detecting both thenon-chemically modified substance and the chemically modified substance.

In the chromatographic process, the method used in chemically modifyingthe substances represented by formulae (1) to (6) and/or theirderivatives at their functional group and subsequently detecting thechemically modified substances is not particularly limited, and in anexemplary method, the substance collected from the armpit and thereagent for chemical modification (hereinafter also referred to as thechemical modification reagent) may be reacted for chemical modificationand the chemically modified substance may be detected after thechromatographic separation (This method is hereinafter also referred toas the “pre-column modification”). Alternatively, the substancesrepresented by formulae (1) to (6) and/or their derivatives in thesubstance collected from the armpit can be separated by achromatographic process, and then, a chemical modification reagent maybe added for chemical modification to thereby enable detection of thechemically modified substance (This method is hereinafter also referredto as the “post-column modification”). Such pre-column modification andpost-column modification may also be used in combination.

The method used in the chromatographic process to chemically modify thesubstances represented by formulae (1) to (6) and/or their derivativesin order to improve the detection sensitivity, is not particularlylimited. However, exemplary modifications which may be used in theliquid chromatographic process include incorporation of a chromophore (acompound exhibiting absorption for light in visible and UV range andcompound having the character of emitting fluorescence) which improvesdetection sensitivity in at least one of the functional groups (forexample, amino group, carboxy group, hydroxy group, carbonyl group,thiol group, and amide group) in the molecular structure of thesubstances represented by formulae (1) to (6) and/or their derivatives,and use of chemiluminescence and electrochemical activity. Exemplarymodifications which may be used in the liquid chromatographic processinclude derivatization of the substance to a compound vaporizable in thegiven temperature.

The method used in incorporating an atomic group in at least one of thefunctional groups (for example, an amino group, carboxy group, hydroxygroup, carbonyl group, thiol group, and amide group) in the molecularstructure of the substances represented by formulae (1) to (6) and/ortheir derivatives for improving the detection sensitivity is notparticularly limited. In an exemplary method, a chromophore forimproving the detection sensitivity is incorporated by means of variouschemical reactions.

More specifically, a typical example in which a chromophore whichabsorbs visible or UV light or a chromophore which emits fluorescenceincorporated in the amino group, carboxy group, or hydroxy group of thesubstance represented by formula (1) is the substance represented by thefollowing formula (8).

In formula 8, R¹, R², and R³ are as defined above. X₁ shows typicalexamples in which a chromophore has been introduced in the amino groupof the substance represented by formula (1); X₂ shows typical examplesin which a chromophore has been introduced in a carboxy group of thesubstance represented by formula (1); and X₃ shows typical examples inwhich a chromophore has been introduced in a hydroxy group of thesubstance represented by formula (1).

For example, the amino group of the substance represented by formula (1)may be converted into other forms such as sulfonamide, carboxamide,thiourea, or amine. Similarly, the carboxy group of the substancerepresented by formula (1) may be converted to other forms such asester, isourea, urea, or other forms. The hydroxy group may be convertedto ether, urethane, ester, or other forms.

In incorporating a chromophore exhibiting absorption for visible and UVlight or a chromophore having the character of emitting fluorescencewhich improves detection sensitivity in at least one of the functionalgroups (for example, amino group, carboxy group, hydroxy group, carbonylgroup, thiol group, and amide group) in the molecular structure of thesubstances represented by formulae (1) to (6) and/or their derivatives,the chromophore used may be, for example, nitrophenol, dinitrophenol,azobenzene, coumarin, 2,1,3-benzoxadiazole, naphthalene, acridine,fluorescein, an analogue thereof, or other chromophore although thechromophore used is not particularly limited, and the functional groupmay be converted to the substituents X₁ to X₃ shown in formula (8) or ananalog thereof.

The method used in incorporating a chromophore which improves detectionsensitivity in at least one of the functional groups in the molecularstructure of the substances represented by formulae (1) to (6) and/ortheir derivatives using various chemical reactions is not particularlylimited, and such incorporation may be accomplished either by using acommercially available chemical modifier (derivatizing agent) or byfreshly synthesizing a chemical modifier which can be used for thesubstances represented by formulae (1) to (6) and/or their derivativesas desired for use in the chemical modification.

Next, typical commercially available chemical modifiers (also referredto as the derivatizing agents) which can be used in the presentinvention is described.

Exemplary reagents which may be used in incorporating a (visible lightand/or UV absorbing) chromophore in the amino group of the substancesrepresented by formulae (1) to (6) and/or their derivatives include (a)an acyl chloride, (b) an allyl sulfonyl chloride, (c) a nitrobenzene,and (d) an isocyanate or isothiocyanate.

Exemplary acyl chlorides (a) include p-methoxybenzoyl chloride,m-toluoyl chloride, p-nitrobenzoyl chloride, and benzoyl chloride, andexemplary allyl sulfonyl chlorides (b) include toluene sulfonyl chloride(TSCl), benzenesulfonyl chloride (BSCl), and dimethylaminoazobenzenesulfonyl chloride (DABSCl).

Exemplary nitrobenzenes (c) include 1-fluoro-2,4-dinitrobenzene (FDNB),trinitrobenzenesulphonic acid (TNBS), and4-fluoro-3-nitrobenzotrifluoride (FNBT), and exemplary isocyanates orisothiocyanates (d) include phenyl isocyanate (PIC), naphthyl isocyanate(NIC), phenyl isothiocyanate (PITC), naphthyl isothiocyanate (NITC),4-N,N′-dimethylamino azobenzene-4′-isothiocyanate, and p-phenyl benzoylisothiocyanate.

Other examples include p-nitro benzyl bromide (p-NBBr), dansyl chloride(Dns-Cl), o-phthaldialdehyde (OPA), ninhydrin, and 1,2,3-perinaphthindantrione (Peri).

Exemplary reagents which may be used in incorporating a (fluorescent)chromophore in the amino group of the substances represented by formulae(1) to (6) and/or their derivatives include (a) sulfonyl chloride, (b)carbonyl chloride, (c) halogeno nitro benzofuran, (d) isocyanate orisothiocyanate, (e) Schiff base generating reagent and its relatedreagents.

Exemplary sulfonyl chlorides (a) include 5-dimethylaminonaphthalene-l-sulfonyl chloride (Dns-Cl), 5-di-n-butylaminonaphthalene-1-sulfonyl chloride (BNS-Cl), 6-N-methyl anilinonaphthalene-2-sulfonyl chloride (Mns-Cl),2-p-chlorosulfophenyl-3-phenylindone (Dis-Cl), 1,2-naphthalenebenzimidazole-6-sulfonyl chloride, and 8-methoxy quinoline-5-sulfonylchloride.

Exemplary carbonyl chlorides (b) include 9-fluorenylchloro methylformate (FMOC), 2-naphthyl chloroformic acid (NCF), 2-dansylchloroethylformate, and (R,S)-2-(p-chlorophenyl)-α-methyl-5-benzoxazole acetylchloride, and exemplary halogeno nitrobenzofurans (c) include4-chloro-7-nitro-2,1,3-benzoxadiazole (4-chloro-7-nitro benzofurazan;NBD-Cl), and 4-fluoro-7-nitro benzofurazan (NBD-F).

Exemplary isocyanates or isothiocyanates (d) include 9-isothiocyanateacridine, fluorescein isothiocyanate,4-dimethylamino-1-naphthylisothiocyanate, 4-(benzyloxycarbonylaminomethyl)phenylisothiocyanate, and 4-(dimethylaminonaphthalene-1-sulfonylamino)phenylisothiocyanate.

Exemplary Schiff base generating reagents and its related reagents (e)include pyridoxal and pyridoxal phosphoric acid, 2-fluorenecarboxyaldehyde, 1-pyrene carboxyaldehyde, o-phthaldialdehyde/alkylthiol(OPA/R-SH) reagent, 2-acetyl benzaldehyde/ethanethiol (OAB),ω-formyl-o-hydroxyacetophenone, and benzo-γ-pyrone, benzoin(2-hydroxy-2-phenylacetophenone).

Also included are 4-phenylspiro[furan-2(3H),1′-phthalan]-3,3′-dione(fluorescamine), 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF),N-succinimidyl-2-naphthoxyacetate, and N-succinimidyl-1-naphthylcarbamate, 5-(4,6-dichloro-1,3,5-triazine-2-yl)aminofluorescein (DTAF).

Exemplary reagents which may be used in incorporating a (visible lightand/or UV absorbing) chromophore in the carboxy group of the substancesrepresented by formulae (1) to (6) and/or their derivatives include (a)phenacyl bromide, naphthacyl bromide, and their related compound, and(b) N-methylphthalimide derivative.

Exemplary phenacyl bromides, naphthacyl bromides, and their relatedcompounds (a) include p-bromophenacyl bromide, phenacyl bromide,naphthacyl bromide, p-nitrophenacyl bromide, and1-(4-hydroxyphenyl)-2-bromoethanone (4-HBE), and exemplary N-methylphthalimidederivatives (b) include N-chloromethyl phthalimide (CIMPI),N-chloromethyl-4-nitro phthalimide (CIMNPI), and N-chloromethylisatin(CIMIS).

Also included are O-p-nitrobenzyl-N,N′-diisopropyl isourea (p-NBDI),2-nitrophenylhydrazine, o-phenylenediamine, pyridinium dichromate,2-methyl quinoxanol derivative, and imidazole.

Exemplary reagents which may be used in incorporating a (fluorescent)chromophore in the carboxy group of the substances represented byformulae (1) to (6) and/or their derivatives include fluorescentcoumarin derivatives.

Exemplary fluorescent coumarin derivatives include4-bromomethyl-7-methoxycoumarin (Br-Mmc),4-bromomethyl-6,7-dimethoxycoumarin (Br-Mdmc),4-bromomethyl-7-acetoxycoumarin (Br-Mac),4-diazomethyl-7-methoxycoumarin,N,N′-dicyclohexyl-O-(7-methoxycoumarin-4-yl)methylisourea,N,N′-diisopropyl-O-(7-methoxycoumarin-4-yl)methylisourea, 9-bromomethylacridine, 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxaline,naphthacyl bromide(2-bromoacetonaphthone), p-(anthroyloxy)phenacylbromide (panacyl bromide), 1-bromoacetylpyrene,9-chloromethylanthracene, and 9-anthryl diazomethane (ADAM).

Alternatively, the carboxy group of the substances represented byformulae (1) to (6) and/or their derivatives may be activated with areagent such as oxalyl chloride, N-ethyl-N′-(3-dimethyl aminopropyl)carbodiimide, N,N′-carbonyldiimidazole, or 2-bromo-l-methylpyridiniumiodide, and reacted with an alcohol or an amine.

For example, the substances represented by formulae (1), (3), and (4)and/or their derivatives may be reacted with oxalyl chloride to producean acid chloride, and the acid chloride may be reacted with9-aminophenanthrene or 1-naphthylamine in the presence of triethylamineto derivatize an amide compound which emits strong fluorescence.

Exemplary alcohols suitable for use in labeling an activated carboxylicacid with fluorescence include 9-hydroxymethyl anthracene,2-dancylaminoethanol, and 4-hydroxymethyl-7-methoxy coumarin (HO-Mmc),and a fluorescent ester compound may be derivatized by using such analcohol.

Exemplary reagents which may be used in incorporating a (visible lightand/or UV absorbing) chromophore or an atomic group containing suchchromophore in the hydroxy group of the substances represented byformulae (1) to (6) and/or their derivatives include an acyl chloride,phenyl isocyanate (PIC), and phenyldimethylsilyl chloride.

Exemplary acyl chlorides include benzoyl chloride, p-methoxybenzoylchloride, p-nitrobenzoyl chloride, 3,5-dinitrobenzoyl chloride, andanthracene-9-carbonyl chloride.

Exemplary reagents which may be used in incorporating a (fluorescent)chromophore in the hydroxy group of the substances represented byformulae (1) to (6) and/or their derivatives include7-methoxycoumarin-3-carbonyl azide (3-MCCA),7-methoxycoumarin-4-carbonyl azide (4-MCCA), 7-(chlorocarbonylmethoxy)-4-methylcoumarin, 4-dimethylamino-l-naphthoyl nitrile,1-anthroyl nitrile, 9-anthroyl nitrile,2-methyl-1,1′-binaphthalene-2′-carbonyl nitrile, naphthalene boric acid,and phenanthrene boric acid.

Exemplary reagents which may be used in incorporating a (visible lightand/or UV absorbing, or fluorescent) chromophore in the thiol group ofthe substances represented by formulae (1) to (6) and/or theirderivatives include N-(9-acridinyl)maleimide (NAM),4-Chloro-7-sulfobenzofurazan, ammonium salt (SBD-Cl),4-fluoro-7-sulfobenzofurazan, ammonium salt (SBD-F),4-fluoro-7-sulfamoyl benzofurazan (ABD-F), N-[4-(5,6-methylenedioxy-2-benzofuranyl)phenyl]maleimide (MBPM),N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM),N-[p-(2-benzimidazolyl)phenyl]maleimide, monobromobimane,5,5-dithiobis(2-nitro benzoic acid), phenazine methosulfate, ando-phthalaldehyde/2-aminoethanol.

Exemplary chemical modification methods which may be used inincorporating a (visible light and/or UV absorbing) chromophore in theamido group of the substances represented by formulae (1) to (6) and/ortheir derivatives include color development by hydroxamic acid-ironchelate and color development of N-arylamide by azo dye generation.

Liquid chromatography is a process wherein the solute is separated bydifference in the affinity for the stationary phase and the mobilephase, and the present invention can make use of various separationmechanisms (interaction between the elutes and the stationary phase).Examples include adsorption chromatography, partition chromatography,ion exchange chromatography, and size exclusion chromatography (gelfiltration chromatography or gel permeation chromatography).

When the substances represented by formulae (1) to (6) is detected bygas chromatography and/or gas chromatography-mass spectrometer, at leastone of the functional groups in the molecular structure of thesubstances represented by formulae (1) to (6) and/or their derivativesmay be chemically modified based on various chemical reactions mainlyfor the purposes of imparting volatility, improving detectionsensitivity, and improving resolution. The reagents suitable for suchpurposes are not particularly limited, and any suitable reagent may beused to enable the detection of the chemically modified substancesrepresented by formulae (1) to (6) and/or their derivatives.

In an exemplary process, the substance represented by formula (1) may beimparted with volatility by esterifying the carboxy group of thesubstance represented by formula (1), and acylating the amino group ofthe substance represented by formula (1) for conversion into anN-acylalkyl ester which is a volatile derivative.

More specifically, examples of the alkyl ester in the above-mentionedN-acylalkyl ester include a methyl group, ethyl group, propyl group,n-and isobutyl group, and n- and isoamyl group. Exemplary methods forthe methyl ester synthesis include a method using diazomethane or borontrifluoride with methanol, and a method using trimethylsilyldiazomethane. Examples of the N-acyl group in the above-mentionedN-acylalkyl ester include an acetyl group, trifluoroacetyl group,pentafluoro propionyl group, and heptafluoro butyryl group. Examples ofa method for introducing N-acyl group include a method in whichanhydrous trifluoroacetic acid, trifluroacetic acid imidazole or thelike is used.

Alternatively, the substance represented by formula (1) may be impartedwith volatility by trimethylsilylating the carboxy group and/or theamino group in the substance represented by formula (1) to produce theN-trimethylsilylated form of the substance which is a volatilederivative.

Exemplary reagents which may be used for the trimethylsilylation includeN-trimethylsilylimidazole (TMSI),N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA),N,O-bis(trimethylsilyl)acetamide (BSA),N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), N-trimethylsilyldimethylamine (TMSDMA), N-methyl-N-trimethylsilyl-acetamide (MTMSA), andhexamethyldisilazane (HMDS).

The substance represented by formula (1) may also be imparted withvolatility by esterifying the carboxy group of the substance representedby formula (1) by using methanol, ethanol, or butanol with hydrochloricacid, and trimethylsilylating the amino group of the substancerepresented by formula (1) for conversion into an N-trimethylsilylaminoacid ester which is a volatile derivative.

In the present invention, determination of the body odor may also beconducted by detecting a substance known to be responsible for the bodyodor, for example, androstenones such as androstenone (5

-16-androstene-3-one), androstenol (5

-16-androstene-3

-ol or 5

-16-androstene-3

-ol), androstadienone (4,16-androstadiene-3-one) and/or its derivative;a lower aliphatic carboxylic acid such as acetic acid, propionic acid,butyric acid, isobutyric acid, valeric acid, isovaleric acid, 4-ethylheptanoic acid, or 7-octenoid acid; aldehydes and ketones such as1-octene-3-one or cis-1,5-octadiene-3-one, in addition to the substancesrepresented by formulae (1) to (6).

Immunochemical assay (also referred to as the “immunoassay”) is an assayin which the amount of an antigen is chemically measured by means of animmunological reaction which is a means used in the body system forrecognizing a molecule, namely, by the function (“antigen-antibodyreaction”) of capturing a particular substance called “antigen” by an“antibody” at a high sensitivity and a high selectivity to therebyinhibit the action of the antigen. In the present invention, suchimmunochemical assay may also be applied for detecting the substancesrepresented by formulae (1) to (6) and/or their derivatives at a highsensitivity and a high selectivity.

In preparing an antibody against the substances represented by formulae(1) to (6), a spacer molecule or the like may be introduced as desiredin the substances represented by formulae (1) to (6) to thereby increasetheir molecular weight before preparing the antibody.

The method used in detecting the amount of the substances represented byformulae (1) to (6) and/or their derivative in the sample collected fromthe armpit is not particularly limited. However, the substance detectedmay be labeled to realize a detection at a higher sensitivity. Suchlabeling may be accomplished, for example, using a radioisotope(radioimmunoassay), an enzyme (enzyme immunoassay), or a fluorescent dyeor a chemiluminescent reagent.

In the present invention, the substances represented by formulae (1) to(6) can be detected by a wide variety of methods as described above, andwhen a reagent having an atomic group which exhibits absorption in thevisible range (hereinafter also referred to as a “color reagent”) isused as a chromophore, the color developed may be detected either with avisible-UV spectrophotometer or with the naked eye.

When the substances represented by formulae (1) to (6) are detected withthe naked eye, the degree of the apocrine odor may be determined bypreparing a color development standard sample indicating the relationbetween the color developed and the concentration of the labeledcompound, and comparing the sample collected from the armpit with thethus prepared color development standard. For example, the substance ofinterest may be reacted with the color reagent or the like and theresulting aqueous solution may be filled in colorimetric tubes orimpregnated in filter papers to thereby prepare color developmentstandard samples, and the body odor may be determined by evaluating thecolor change of the substance collected from the armpit upon reactionwith the color reagent by comparing with the color development standardsample. The determination of the body odor is thereby enabled.Alternatively, it is also possible to conduct the determination bydirect evaluation of the color of the aqueous solution, or by directevaluation of the color of the test paper having the aqueous solutionimpregnated therein with the naked eye.

When the method as illustrated above is used, the amount of thesubstances represented by formulae (1) to (6) in the armpit can bequalitatively and/or quantitatively assayed, and an objective andquantitative determination of the possibility (potential) of the armpitsmell, and in particular, the apocrine odor generation is enabled on thebases of the assay results.

Under the conditions when a large amount of the substances representedby formulae (1) to (6) are generated with the apocrine odor not yetgenerated, the apocrine odor can be deemed “latent”. In such “latent”situation, an objective and quantitative determination of the body odoris not possible even if an assay using the component responsible for theapocrine odor directly for the index were conducted.

In contrast, the substance detected in the present invention is theprecursor before turning into the main substance responsible for theapocrine odor, and therefore, an objective and quantitativedetermination of whether the test subject is inclined to generateapocrine odor or not, namely, the possibility (potential) of theapocrine odor generation is thereby enabled.

The present invention has enabled a highly objective determination byusing the substances represented by formulae (1) to (6) for the index,and also, a quantitative determination is realized by interpreting theassay data as the amount of the substance. The present invention is alsocapable of determining the effectiveness of the deodorant which isexpected to have preventive action for the apocrine odor generation bydetecting the substances represented by formulae (1) to (6).

For example, the substance represented by formula (2) which is thesubstance mainly responsible for the apocrine odor is a substanceproduced by the change of the substance represented by formula (1) bythe action of, for example, a microorganism on the skin, and therefore,an objective and quantitative determination of the effectiveness of thedeodorant sample which is expected to have a preventive action for theapocrine odor can be accomplished by measuring the substance representedby formula (1) and/or the substance represented by formula (2).

More specifically, change in the amount of the substance represented byformula (1) and/or the substance represented by formula (2) can becompared by detecting the substance represented by formula (1) and/orthe substance represented by formula (2) before and after applying theanalyte which is expected to have the preventive action for the apocrineodor generation. This enables an objective and quantitativedetermination of the effect of the analyte in preventing the change ofthe substance represented by formula (1) to the substance represented byformula (2), namely, the effect of the analyte in preventing theapocrine odor generation.

In addition, since the substances represented by formulae (5) and (6)which are the main substances responsible for the apocrine odor are thesubstances respectively produced by the change of the substancesrepresented by formulae (3) and (4) by the action of, for example,microorganisms on the skin, further detection of the substancesrepresented by formulae (3) and (4) and/or the substances represented byformulae (5) and (6) enables determination of the effectiveness of thedeodorant sample which is expected to have a preventive action on theapocrine odor generation at an improved objectivity and quantitativeaccuracy.

More specifically, change in the amount of the substances represented byformulae (3) and (4) and/or the substances represented by formulae (5)and (6) can be compared by detecting the substances represented byformulae (3) and (4) and/or the substances represented by formulae (5)and (6) before and after applying the analyte which is expected to havethe preventive action for the apocrine odor generation. This enables anobjective and quantitative determination of the effect of the analyte inpreventing the change of the substances represented by formulae (3) and(4) to the substances represented by formulae (5) and (6), namely, theeffect of the analyte in preventing the apocrine odor generation at animproved objectivity and quantitative accuracy.

In actually determining the effectivity of the deodorant sample which isexpected to prevent the apocrine odor generation, the determination maybe conducted by actually applying the analyte to the human armpit,collecting samples from the armpit before and after the analyteapplication, and detecting the substances represented by formulae (1) to(6) in the collected samples to thereby determine the effectiveness ofthe analyte.

In determining effectiveness of the deodorant sample which is expectedto prevent generation of the apocrine odor, the substances representedby formulae (1) to (6) may be used either alone or in combination of twoor more. In addition, the deodorant sample may also contain additivesnormally incorporated in an antiperspirant deodorant, for example, asolvent, stabilizer, antiperspirant, bactericide, antibacterial agent,surfactant, antioxidant, flavor, plant and extract.

EXAMPLES Synthesis of1.2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid (formula (9))

206 mg (1.39 mmol) of (S)-3-mercapto-3-methyl-hexane-1-ol and anequimolar amount of N-α-t-butoxycarbonyl-3-chloro-L-alanine(manufactured by Watanabe Chemical Industries, Ltd.) were dissolved in 4mL of N,N-dimethylformamide, and to this solution, 2.6 equivalents ofNaH (sodium hydride) was added in several aliquots while payingattention to heat generation and foaming. After completing the reaction,diethylether and ice water were added for phase separation. Ethylacetate was added to the aqueous layer, and dilute hydrochloric acid wasadded for precipitation. After separation, the aqueous layer was againextracted with ethyl acetate. The ethyl acetate layers were combined,washed with water and saturated aqueous solution of sodium chloride, anddried for concentration to thereby obtain an oil.

Next, the target compound was purified by separation by HPLC on ODScolumn using MeCN/water/0.1% trifluoroacetic acid for the elutingsolution. The eluted fractions of the target compound were combined, andafter neutralization, MeCN was removed by distillation. After addingdilute hydrochloric acid and extracting with ethyl acetate, the solutionwas washed with water, and then, with saturated aqueous solution ofsodium chloride. To the oil obtained by drying and concentration, 400 μLof 4N HCl/dioxane was added. The concentrate was again purified byseparation by HPLC on ODS column using MeCN/water/0.1% trifluoroaceticacid for the eluting solution. The eluted fractions of the targetcompound were freeze dried to obtain 38 mg of viscous oil.

The synthesized product was evaluated for NMR spectrum by recording withMercury 400Vx (manufactured by Varian). Chemical shift δ is shown belowin ppm in relation to TMS which was used for the reference substance.The MS spectrum data recorded by LC-MS 2690 (manufactured by Waters) isalso shown below. The NMR and MS spectrum obtained supported thestructure of the target compound.

¹H-NMR (DMSO): 0.894 (t,CH₃), 1.250 (s, CH₃), 1.358 (m, CH₂), 1.447 (m,CH₂), 1.671 (t, CH₂), 2.881 (m, (S—)CH₂), 3.519 (t, CH₂—OH), 4.067 (t,CH—NH₂)

MS: m/z=226 (Ml⁺¹, relative strength 100%), m/z=122 (relative strength33%)

2. Analysis of Apocrine Odor and its Precursor in Human Armpit

(1) Analysis of Apocrine Odor

65 healthy Japanese males were randomly selected for the test subjects,and they continuously wore new white cotton T-shirt for 24 hours. TheT-shirt was collected, and the part that had been in contact with thearmpit was used for sensory evaluation by a panel of 5 specialists (3males and 2 females) for assessment of the type and the strength of thearmpit smell.

<Items Evaluated in Determining the Armpit Smell Type>

Apocrine odor: a stinky smell with the smell like soy sauce orgrapefruit

Acid odor (lower fatty acid odor): sour putrid smell

<Criteria for Strength of the Armpit Smell>

0: No smell

1: Slight smell

2: Weak smell

3: Moderate smell

4: Considerable smell

5: Strong smell

As a result, 28 people had little armpit smell (with the score of thearmpit smell strength of 1 or less) (group A), 21 people had weak armpitsmell (with the score of the armpit smell strength of 2 or 3) (group B),and 16 people had strong armpit smell (with the score of the armpitsmell strength of 4 or 5) (group C).

(2) Analysis of Apocrine Odor Precursor

The test subjects of this analysis were 3 subjects of group C who hadstrong apocrine odor and 3 subjects of group C who had no apocrine odor.About 2 mL of sweat was collected in a test tube from the armpit on bothsides of each test subject in a room adjusted to a temperature of 40° C.and a humidity of 80%. The sweat (of each of the 6 samples) was filteredthrough cotton, and then, through a sterilization filter (0.2 μm), and0.05N HCl aqueous solution was added to adjust the pH to 3.3. Next, thesweat was washed with distilled ether (0.4 mL×5 times), and then, withhexane (0.4 mL×5 times) to remove the oil soluble content. The residualsolvent was removed by distillation using a rotary evaporator at roomtemperature for several hours. After filtration through thesterilization filter (0.22 μm), the solution was subjected toultrafiltration. When the fraction having a molecular weight of up to5000 was freeze dried, a transparent to slightly whitish viscous solidwith no odor was obtained. All samples were stored at −40° C. untilimmediately before the analysis.

The samples prepared as described above were analyzed by HPLC-MS/MS massspectroscopy.

<Measurement Conditions>

HPLC system: Agilent Model 1100 (manufactured by Agilent Technologies)

Analysis column: Inertsil ODS-3 (2.1 mm ID×250 mm)

Mobile phase: 50% (1% aqueous solution of acetic acid)/50% (1% methanolsolution of acetic acid)

Mass spectrometer (MS/MS): API 4000 (Applied Biosystems/MDS Sciex)

The results of the analysis reveal that2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid(formula (9))was present in the sweat of the test subject having apocrine odor asshown in FIG. 1 (FIGS. 1 a to 1 c) and FIG. 2 (FIGS. 2 a to 2 c).

The results of the analysis reveal that2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid(formula (9))was absent in the sweat of the test subject having no apocrine odor asshown in FIG. 3 (FIGS. 3 a and 3 b).

(3) Analysis of the Mechanism of Apocrine Odor Generation

The test subjects of this analysis were 3 subjects of group C withconsiderable apocrine odor (with the score of the armpit smell strengthof 4). 0.1 mg of 2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoicacid(synthetic product, formula (9)) was dissolved in 0.1 mL of distilledwater, and one armpit of the test subject was sprayed with this aqueoussolution. The other armpit was sprayed with 0.1 mL of distilled water.After 8 hours, 5 expert panelists (3 males and 2 females) evaluated thearmpit odor by sensory test. All of the expert panelists then answeredthat the stinky beastly smell like that of sulfur from3-mercapto-3-methyl-hexane-1-ol was significantly stronger in the armpitof the side sprayed with the aqueous solution containing2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid (syntheticproduct, formula (9)) compared to the armpit of the side sprayed onlywith the distilled water. In other words, it was demonstrated that2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid decomposes intothe 3-mercapto-3-methylhexane-1-ol on the skin of the human armpit.

3. Evaluation of Effectiveness of the Deodorant for Apocrine Odor

The test subject was one healthy American female in her thirties. Thetest subject was prohibited from using an antiperspirant deodorant orthe like in the armpit from 10 days before the test. On the morning ofthe test, after confirming that the armpit odor was at the same level onboth sides, 0.3 g of the deodorant (stick type) was applied to onearmpit. After 10 hours, sweat from both armpits was collected by wipingwith a cotton gauze (5 cm×8 cm). The cotton gauze was divided in 8 equalpieces, fully wetted with 6 mL of distilled water, and the liquidabsorbed in the gauze was squeezed for collection with a syringe. Thegauze was wetted again with 6 mL of distilled water, and collected bysqueezing with a syringe. After centrifugation, the extracts werecombined, and washed twice with hexane (10 mL). The solvent was removedby distillation, and the residue was freeze dried, dissolved with mobilephase used for the HPLC, and subjected to ultrafiltration to collect thetest sample having a molecular weight of up to 5,000. Standard solutionsof different concentrations were separately prepared using2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid. First, thestandard solutions were analyzed by LC-MS/MS to depict a calibrationcurve of the 2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid(synthetic product). Next, the test samples were analyzed by LC-MS/MS tomeasure the amount of2-amino-7-hydroxy-5-methyl-5-propyl-4-thiaheptanoic acid, namely, theprecursor of the present invention, in the test samples. Rate ofdeodorization was thereby calculated.

<Measurement Conditions>

HPLC system: LC-10ADvp (manufactured by Shimadzu Corporation)

Analysis column: Inertsil ODS-3 (2.1 mm ID×250 mm)

Mobile phase: 50% (1% aqueous solution of acetic acid)/50% (1% methanolsolution of acetic acid)

Mass spectrograph (MS/MS): API 2000 (Applied Biosystems/MDS Sciex) Q1/Q3masses=236.10/122.00 amu

<Deodorant formulation> (Ingredient) (% by weight) Stearyl alcohol 16Behenyl alcohol 0.5 Hardened castor oil 3.5 Decamethylcyclopentasiloxane 43 Aluminum zirconium tetrachlorohydrex glycinecomplex 24 Talc 7 Fragrance 1 Others 5 Total 100

As a result of the analysis, effectiveness of the deodorant (stick type)which was expected to suppress the generation of the apocrine odor wasconfirmed as shown in Table 1.

TABLE 1 2-amino-7-hydroxy- 5-methyl-5-propyl- 4-thiaheptanoic acid (thevalue Deodoration measured, ng) rate (%)*1 Right armpit Application of0.3 g 44.4 73.4 of the deodorant Left armpit Not treated 167.1*1Deodoration rate (%) = [1 − {(the value measured for the side appliedwith the deodorant)/(the value measured for the non-treatment side)}] ×100

1. A compound of formula (1a):


2. A method for determining potential for body odor or latent apocrineodor carried out by detecting a compound of formula (1a):


3. The method of claim 2 comprising determining potential for body odor.4. The method of claim 2 comprising determining potential for latentapocrine odor.
 5. The method of claim 2, comprising detecting thepresence or absence of a potential for body odor or latent apocrine odorby quantitatively detecting said compound.
 6. The method of claim 2,comprising quantitatively determining the amount of said compound. 7.The method of claim 2, comprising quantitatively determining the amountof said compound and determining a greater potential for body odor orlatent apocrine odor in a sample having a higher level of said compoundthan in a control sample.
 8. The method of claim 2, further comprisingcollecting a sample of sweat from a subject or collecting a sample fromthe armpit of from a subject.
 9. The method of claim 2, furthercomprising measuring the conversion of said compound into a compound offormula (2a) 3-mercapto-3-methylhexane-1-ol.
 10. The method of claim 2,comprising detecting said compound by chromatographic separation andassay.
 11. The method of claim 2, further comprising detecting theability of a candidate deodorant to reduce the amount of said compoundin a sample or to decrease the amount of the compound formula (2a)3-mercapto-3-methylhexane-1-ol in a sample.